CN102085226B - Japanese picris japonica extract and extracting method thereof - Google Patents
Japanese picris japonica extract and extracting method thereof Download PDFInfo
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- CN102085226B CN102085226B CN2011100241786A CN201110024178A CN102085226B CN 102085226 B CN102085226 B CN 102085226B CN 2011100241786 A CN2011100241786 A CN 2011100241786A CN 201110024178 A CN201110024178 A CN 201110024178A CN 102085226 B CN102085226 B CN 102085226B
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Abstract
The invention discloses a Japanese picris japonica extract and an extracting method thereof. The extractive comprises a water elution part of a water extract of the Japanese picris japonica and total flavone. The extracting method of the water elution part of the water extract of the Japanese picris japonica comprises the following steps of: adding distilled water of a quantity of 15-20 times of the Japanese picris japonica to extract the Japanese picris japonica; combining extracting solutions, centrifuging and taking the supernate; concentrating to a concentration of 1g raw medicinal material per ml, separating and eluting through resin; and concentrating the eluent. The extracting method of the total flavone of the Japanese picris japonica comprises the following steps of: extracting the Japanese picris japonica with 30-60 percent alcohol of a quantity of 20-30 times; centrifuging and then concentrating the extracting solution until no alcohol residue; diluting to a concentration of 1g raw medicinal material per ml with water and then depositing off polysaccharide with 95 percent alcohol of a quantity of 3-5 times; taking the supernate, concentrating and evaporating to dryness; dissolving with water until the concentration of flavone is 20-30mg/ml; separating through resin, and eluting respectively with water and alcohol; and remaining the alcohol eluting part. The Japanese picris japonica extract can be used for medicaments for reducing blood sugar.
Description
Technical field
The present invention relates to a kind of extract and method for distilling thereof of Picris japonica Thunb.
Background technology
The Fols Picridis fuscipilosae platymiscium comprises Picris japonica Thunb, Fols Picridis fuscipilosae (former subspecies Fols Picridis fuscipilosae, single mao of Fols Picridis fuscipilosae), Picris divaricata, Xinjiang Fols Picridis fuscipilosae, annual, life in 2 years, perennial branch draft.
Picris japonica Thunb (
Picris japonciaThunb) be feverfew, have another name called: rifle cutter dish.Mainly be distributed in ground such as Hebei, Shanxi, Shaanxi, Jilin, Heilungkiang in China, wherein Shanxi mainly is distributed in Taiyuan and peripheral counties and cities, Jiao cheng City, and five are waited the area.Be born in patana, the shrubbery or limit, field, river bank, 650～3650 meters of height above sea level.Also there are distribution in Japan and Russia.Herb is gone into mongolian medicine, has heat clearing away, detumescence and analgesic effect.Also there is not report at present about the hypoglycemic activity of Picris japonica Thunb extract.
Summary of the invention
The purpose of this invention is to provide a kind of hypoglycemic activity Picris japonica Thunb extract that has, the method for distilling of this extract is provided simultaneously.
For realizing above-mentioned purpose, a kind of Picris japonica Thunb extract of the present invention comprises the water extract water elution part and the total flavones of Picris japonica Thunb.
Water extract water elution position mainly comprises saccharide compound and flavone compound; Its paste-forming rate is that the 16g thick paste/more than the 100g raw medicinal herbs, polyoses content accounts for more than 30%, and flavones content accounts for more than 5%; The obvious functions of blood sugar effect is arranged, normal mouse blood sugar value there is not influence.General flavone content accounts for more than 50% in the Picris japonica Thunb extractive of general flavone, and the obvious functions of blood sugar effect is arranged, and normal mouse blood sugar value there is not influence.The present invention discloses the hypoglycemic activity of Picris japonica Thunb extract first; It is innocuous substance for the toxicological experiment proof; And further screen the hypoglycemic effective site of Picris japonica Thunb, finally confirm as 2 effective sites of Picris japonica Thunb water extract water elution component and Picris japonica Thunb total flavones.
The method for distilling of the water extract water elution part of Picris japonica Thunb is: Picris japonica Thunb is cleaned, dried, be cut into even segment; Add an amount of distilled water immersion, add distilled water extraction 2-3 time that 15-20 doubly measures, each 2-3h; Extracting solution merges, get supernatant after centrifugal, concentrates, be concentrated into 1g raw medicinal herbs/ml after; Separate through 20 times of amount macroporous adsorbent resins, with 3-4 times of column volume distilled water eluting, concentrate eluant promptly gets.Method for distilling is that reflux, extract,, warm macerating extract or supersound extraction.The process that the Picris japonica Thunb raw medicinal herbs cleans will adopt running water to get express developed, do not soak, in order to avoid lose water miscible composition.
The method for distilling of Picris japonica Thunb total flavones is: Picris japonica Thunb is cleaned, be cut into even segment, doubly measure ethanol extraction 2-3 time of 30%-60% with 20-30; Each 2-3h, it is residual to be concentrated into no ethanol after extracting solution is centrifugal, removes polysaccharide with 3-5 times of 95% ethanol precipitate with ethanol after being diluted with water to 1g raw medicinal herbs/ml; Get supernatant, concentrate evaporate to dryness, water is dissolved to and contains flavone 20-30mg/ml; Separate through macroporous adsorbent resin; With 2-5 times of column volume water elution, 4-6 times column volume 50%-95% ethanol elution, leave and take the ethanol elution part respectively, promptly get.Method for distilling is that warm macerating extracts or reflux, extract, more than 70 ℃.The process that the Picris japonica Thunb raw medicinal herbs cleans will adopt running water to get express developed, do not soak, in order to avoid lose water miscible composition.
(1) investigation of total flavone extracting process: through concentration of alcohol (30%-95%), extraction time (0.5-3h) and number of times (2-4 time), solid-liquid ratio (10-50 doubly measures), the factors such as temperature (40-90 ℃ and reflux, extract), granularity of extracting are investigated; Set up orthogonal experiment; With the content of flavone and purity as evaluation index; Through range analysis and variance analysis; The final extraction process of confirming is: uses 20-40 doubly to measure determining alcohol and is 30%-60% ethanol extraction 2-3 time, and 2-3h at every turn, the extraction temperature is 70-100 ℃.Concentration of alcohol is bigger to the extraction efficiency influence of total flavones with the extraction temperature, is the emphasis of condition control in big the production, and concentration of alcohol obtains maximum flavone in the 30%-60% extraction; Determining alcohol is excessive or too smallly all can obviously reduce the flavone yield, extracts temperature when reaching 70 ℃, and it is the highest that the flavone yield reaches basically; Be lower than 70 ℃, the flavone yield obviously reduces, when being higher than 70 ℃; The almost constant or reduction slightly of flavone yield; But paste-forming rate obviously increases, for further flavone purification brings very big trouble, so do not advise selecting for ease the method for reflux, extract.
(2) investigation of purifying process:
1) screening of resin: as index, detected D with the adsorbance of resin and resolution factor
101, HPD-450, HPD-700, AB-8, polyamide (14-30 order), finally be asserted D
101Macroporous adsorbent resin.
2) investigation of 95% precipitate with ethanol consumption: investigated 2-5 and doubly measured 95% ethanol precipitate with ethanol and remove polysaccharide; With flavones content and purity as evaluation index; Finally confirming as 3-4 doubly measures 95% ethanol precipitate with ethanol and removes polysaccharide; So both can remove polysaccharide composition as far as possible completely, reduce the loss of flavone again to greatest extent.
3) investigation of water consumption in the gradient elution: as evaluation index, investigated the water consumption of 0.2-8 times of column volume with flavones content and purity, confirmed as the loss that the water consumption of 2-5 times of column volume can discard the monosaccharide composition to greatest extent and reduce flavone.
4) investigation of concentration of alcohol and consumption in the gradient elution: with flavones content and purity as evaluation index; Determining alcohol with 10%-95% carries out eluting respectively; The flavones content and the purity that obtain are the highest more than the column volume for 5 of the ethanol elutions of final definite 50%-75%, and purity can reach more than 50%.
Polysaccharide and content of flavonoids are measured in the Picris japonica Thunb:
The Picris japonica Thunb determination of total flavonoids comprises the steps:
1) preparation of control substance of Rutin solution
Precision takes by weighing the rutin standard substance 18.60mg that is dried to constant weight, the ethanol with 60% (v/v) dissolving, and be transferred to standardize solution in the 100 ml volumetric flasks, and shake up, process the control substance of Rutin solution of 0.186mg/ml.
2) preparation of rutin standard curve
Accurate absorption rutin standard solution 0ml, 0.5ml, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml put respectively in the dry test-tube, respectively add 60 ﹪ ethanol to 5.0ml, add 5% sodium nitrite solution 0.3ml, shake up; Add 10% aluminum nitrate solution 0.3ml behind 6 min, shake up, add 4% sodium hydroxide solution 4ml behind 6 min; Shake up, adding distil water is settled to 10ml, shakes up; Place 15 min, sample blank is made reference, measures its absorbance at wavelength 505nm place.(μ g/ml) is abscissa with concentration C, and absorbance A is that vertical coordinate gets rutin standard curve regression equation: Y=0.0146X-0.0062 (r=0.9991), and the result shows that rutin linear relationship between 9.3～93 μ g/ml is good.
3) Picris japonica Thunb determination of total flavonoids
It is an amount of that precision is measured Picris japonica Thunb total flavones need testing solution, measures its absorbance according to standard curve method colour developing back, and the substitution regression equation calculates general flavone content.
The Picris japonica Thunb determination of polysaccharide comprises the steps:
1) preparation of glucose reference substance solution
Precision takes by weighing 105 ℃ of anhydrous glucose reference substance 0.0104 g that are dried to constant weight, adds the suitable quantity of water dissolving, is transferred in the 100 ml volumetric flasks, adds water to scale, shakes up, and promptly gets the solution of 104 μ g/ml.
2) preparation of glucose directrix curve
Get glucose reference substance solution 0.1 ml, 0.2 ml, 0.4 ml, 0.6 ml; 0.8 ml, each adding distil water to 2.0 ml of 1.0 ml respectively adds phenol 1.0 ml then, shakes up; Drip concentrated sulphuric acid 5.0 ml rapidly, shake up, put heating 15 min in the boiling water bath, be cooled to room temperature then.Measure absorbance at 490 nm places.Concentration with a series of glucose reference substance solution is abscissa, and absorbance is a vertical coordinate, sets up standard curve, and the line retrace of going forward side by side is handled.
Get glucose standard curve regression equation: Y=0.0156x-0.0076 (r=0.9994), glucose linear relationship between 5.2 μ g/ml ~ 52 μ g/ml is good.
3) Picris japonica Thunb determination of polysaccharide
It is an amount of that precision is measured the total need testing solution of Picris japonica Thunb, measures its absorbance according to standard curve method colour developing back, and the substitution regression equation calculates general flavone content.
The establishment of Picris japonica Thunb water extract water elution component blood sugar reducing function, its method is following:
Alloxan causes the foundation of diabetic mice model: the injection volume of alloxan is directly connected to the hypoglycemic effect after blood glucose value after the one-tenth mould rate, mortality rate, Cheng Mo of mice and the administration, raises though dosage can make into the mould rate greatly, and blood glucose value is too high; Mortality rate increases, and can not finish whole administration process, through the investigation to the alloxan consumption; Finally confirm as tail vein injection alloxan 40-60mg/kg, the dosage of 0.1ml/10g becomes the mould rate to reach more than 70%; Blood glucose value is suitable, stable behind the Cheng Mo; Almost do not have death, can accomplish whole experiment, can fully prove the hypoglycemic effect of medicine.
Experiment is divided into groups and administration: press the blood glucose value random packet, (1) laboratory animal: animal is adopted cleaning level Kunming kind white mice, 18-22g, male and female half and half; (2) foundation of diabetes model: adopt tail vein injection alloxan 40-60mg/kg to cause the diabetic mice model; (3) divide into groups and administration: Picris japonica Thunb water extract water elution administration group (0.8-12g raw medicinal herbs/kg), glibenclamide positive drug group (2-5mg/kg), normal group, model group, every day gastric infusion once, successive administration 7-14 days; (4) blood specimen collection and mensuration: fasting 8-10h before the last administration, administration continued fasting 2-4h, the blood sampling of eye socket rear vein beard, 2000-3000r/min is centrifugal to the serum separation, gets serum and measures fasting blood sugar by the glucose kit method; (5) statistical method: total data compares with the t check; Adopt the SPSS11.0 statistical package to carry out statistical analysis, statistical result is with mean+SD (
± s) expression.
5. the establishment of Picris japonica Thunb total flavones blood sugar reducing function, its method is following:
Experiment is divided into groups and administration: be divided into 7 groups at random by blood glucose value: glibenclamide positive controls (2-5mg/kg), metformin positive drug group (300mg/kg), Picris japonica Thunb total flavones (25-400mg/kg), model group, normal group.Successive administration 14d measures the fasting blood sugar that each is organized respectively in 7d and 14d.All the other are the same.
The present invention has obtained 2 effective sites with hypoglycemic activity of Picris japonica Thunb; Has the obvious functions of blood sugar effect; Hypoglycemic effect and Picris japonica Thunb water extract and glibenclamide positive drug group do not have statistical difference (shown in table 1, the table 2); The water solublity of 2 effective sites is all very good, and the purity of total flavones reaches more than 50%, and purity is higher.
Compare with the normal control group: ##P＜0.05; Compare with model control group: * * P＜0.05; Compare with the water extract group: Δ P＜0.05
Compare with the normal control group: ##P＜0.05; Compare with model control group: * * P＜0.05
The specific embodiment
Embodiment 1:
The preparation of Picris japonica Thunb water extract comprises the steps: medical material Picris japonica Thunb 5000g flushing with clean water totally, dries; Be cut into even segment; The distilled water immersion (being advisable) that adds 15 times of amounts with whole immersion medical materials, reflux, extract, 2 times, each 2h; Remove slag, merging filtrate, clarification, filter, be evaporated to thick paste (1g raw medicinal herbs/ml), promptly get the Picris japonica Thunb water extract;
The preparation at Picris japonica Thunb water extract water elution position comprises the steps: that the Picris japonica Thunb water extract is through 20 times of amount D
101Macroporous adsorptive resins separates, and with the distilled water eluting of 3 times of column volumes, concentrate eluant promptly gets Picris japonica Thunb water extract water elution component, gets more than the extractum 700g.
Embodiment 2:
The preparation of Picris japonica Thunb water extract comprises the steps: medical material Picris japonica Thunb 5000g flushing with clean water totally, dries; Be cut into even segment; The distilled water immersion (being advisable) that adds 20 times of amounts with whole immersion medical materials, warm macerating extracts 2 times, each 3h; Remove slag, merging filtrate, clarification, filter, be evaporated to thick paste (1g raw medicinal herbs/ml), promptly get the Picris japonica Thunb water extract;
The preparation at Picris japonica Thunb water extract water elution position comprises the steps: that the Picris japonica Thunb water extract is through 20 times of amount D
101Macroporous adsorptive resins separates, and with the distilled water eluting of 4 times of column volumes, concentrate eluant promptly gets Picris japonica Thunb water extract water elution component, gets more than the extractum 700g.
Embodiment 3:
The preparation of Picris japonica Thunb water extract comprises the steps: medical material Picris japonica Thunb 5000g flushing with clean water totally, dries; Be cut into even segment; The distilled water immersion (being advisable) that adds 18 times of amounts with whole immersion medical materials, supersound extraction 3 times, each 2h; Remove slag, merging filtrate, clarification, filter, be evaporated to thick paste (1g raw medicinal herbs/ml), promptly get the Picris japonica Thunb water extract;
The preparation at Picris japonica Thunb water extract water elution position comprises the steps: that the Picris japonica Thunb water extract is through 20 times of amount D
101Macroporous adsorptive resins separates, and with the distilled water eluting of 5 times of column volumes, concentrate eluant promptly gets Picris japonica Thunb water extract water elution component, gets more than the extractum 700g.
Embodiment 4:
The preparation of Picris japonica Thunb total flavones comprises the steps: Picris japonica Thunb 5000g is cleaned, and is cut into even segment; Using 20 times of amount determining alcohols is 30% alcohol reflux 3 times, each 2h, and extracting temperature is 70 ℃; It is residual to be concentrated into no ethanol after extracting solution is centrifugal, and dilute with water is settled to 5000ml, removes polysaccharide with 5 times of 95% ethanol precipitate with ethanol; Get supernatant, concentrated evaporate to dryness water is dissolved to and contains flavone 20mg/ml, through 20 times of amount D
101Macroporous adsorbent resin is used 5 times of column volume water elutions, 4 times of column volume 50% ethanol elutions respectively, leaves and takes 50% ethanol elution part, gets more than the extractum 140g, gets more than the Picris japonica Thunb total flavones 65g.
Embodiment 5:
The preparation of Picris japonica Thunb total flavones comprises the steps: Picris japonica Thunb 5000g is cleaned, and is cut into even segment; Using 30 times of amount determining alcohols is 60% alcohol reflux 3 times, each 2h, and extracting temperature is 70 ℃; It is residual to be concentrated into no ethanol after extracting solution is centrifugal, and dilute with water is settled to 5000ml, removes polysaccharide with 4 times of 95% ethanol precipitate with ethanol; Get supernatant, concentrated evaporate to dryness water is dissolved to and contains flavone 2530mg/ml, through 20 times of amount D
101Macroporous adsorbent resin is used 2 times of column volume water elutions, 5 times of column volume 50% ethanol elutions respectively, leaves and takes 50% ethanol elution part, gets more than the extractum 140g, gets more than the Picris japonica Thunb total flavones 70g.
Embodiment 6:
The preparation of Picris japonica Thunb total flavones comprises the steps: Picris japonica Thunb 5000g is cleaned, and is cut into even segment; Using 25 times of amount determining alcohols is that 45% ethanol warm macerating extracts 2 times, each 2h, and extracting temperature is 70 ℃; It is residual to be concentrated into no ethanol after extracting solution is centrifugal, and dilute with water is settled to 5000ml, removes polysaccharide with 3 times of 95% ethanol precipitate with ethanol; Get supernatant, concentrated evaporate to dryness water is dissolved to and contains flavone 30mg/ml, through 20 times of amount D
101Macroporous adsorbent resin is used 3.5 times of column volume water elutions, 6 times of column volume 50% ethanol elutions respectively, leaves and takes 50% ethanol elution part, gets more than the extractum 140g, gets more than the Picris japonica Thunb total flavones 75g.
Claims (6)
1. Picris japonica Thunb extract is characterized in that: described extract comprises the water extract water elution part and the total flavones of Picris japonica Thunb;
The method for distilling of the water extract water elution part of Picris japonica Thunb is: Picris japonica Thunb is cleaned, dried, be cut into even segment; Add an amount of distilled water immersion, add distilled water extraction 2-3 time that 15-20 doubly measures, each 2-3h; Extracting solution merges, get supernatant after centrifugal, concentrates, be concentrated into 1g raw medicinal herbs/ml after; Separate through 20 times of amount macroporous adsorbent resins, with 3-5 times of column volume distilled water eluting, concentrate eluant promptly gets;
The method for distilling of Picris japonica Thunb total flavones is: Picris japonica Thunb is cleaned, be cut into even segment, doubly measure ethanol extraction 2-3 time of 30%-60% with 20-30; Each 2-3h, it is residual to be concentrated into no ethanol after extracting solution is centrifugal, removes polysaccharide with 3-5 times of 95% ethanol precipitate with ethanol after being diluted with water to 1g raw medicinal herbs/ml; Get supernatant, concentrate evaporate to dryness, water is dissolved to and contains flavone 20-30mg/ml; Separate through macroporous adsorbent resin; With 2-5 times of column volume water elution, 4-6 times column volume 50%-95% ethanol elution, leave and take the ethanol elution part respectively, promptly get.
2. the method for distilling of Picris japonica Thunb extract according to claim 2 is characterized in that: during the water elution extracting section, method for distilling is that reflux, extract,, warm macerating extract or supersound extraction.
3. the method for distilling of Picris japonica Thunb extract according to claim 2 is characterized in that: when total flavones extracted, method for distilling was that warm macerating extracts or reflux, extract, more than 70 ℃.
4. according to the method for distilling of right 2 or 3 described Picris japonica Thunb extracts, it is characterized in that: during the water elution extracting section, after 20 times of amount macroporous adsorbent resins separated, with 4 times of column volume distilled water eluting, concentrate eluant promptly got.
5. according to the method for distilling of claim 2 or 4 described Picris japonica Thunb extracts, it is characterized in that: when total flavones extracts, after macroporous adsorbent resin separates, use 3.5 times of column volume water elutions, 5 times of column volume 50%-95% ethanol elutions respectively.
6. the application of Picris japonica Thunb extract according to claim 1 in the preparation hypoglycemic drug.
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